1. Field of the Invention
This invention relates to novel recombinant nucleic acids encoding the enzyme dihydrofolate reductase (DHFR) from mycobacteria, to novel recombinant DHFR peptides produced by such sequences, and to vaccines, diagnostic kits, cells and therapies utilizing these peptides and nucleic acid sequences. The invention is also directed to methods for using the sequences and peptides to develop drugs specific to M. avium and other species of mycobacteria, to identifying other DHFR sequences and peptides, as well as diagnostic and treatment methods incorporating the disclosed sequences and peptides.
2. Description of Background
The Mycobacterium avium complex represents one of the most serious opportunistic infections and is often associated with advanced stages of autoimmune deficiency syndrome or AIDS (J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991; J. A. Havlok, Jr. et al., J. Infec. Dis. 165:577-80, 1992; C. C. Hawkins et al., Ann. Intern. Med. 105:184-88, 1986; D. S. O'Brien et al., Am. Rev. Respir. Dis. 135:1007-14, 1989; N. Rastogi et al., Res. Microbiol. 145:167-261, 1994). Unlike Mycobacterium tuberculosis, which can be successfully treated with two or three drug combinations (except for multidrug resistant M. tuberculosis; MDR-TB), the M. avium complex is resistant to many antimycobacterial agents (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intern. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; L. Heifets et al., Antimicrob. Agents Chemother. 37:2364-70, 1993; D. Y. Rosenzweig, Amer. Rev. Resp. Dis. 113(Suppl.):55, 1976). Drug resistance in M. avium is still considered an inherent property of the wild type organism (S. L. Morris et al., Complex. Res. Microbiol. 147:68-73, 1996), resulting in large part from the refractory nature of the organism's cell envelope (H. L. David, Rev. Infect. Dis. 3:878-84, 1981; N. Rastogi et al., Res. Microbiol. 145:243-52, 1994; N. Rastogi et al., Antimicrob. Agents Chemother. 20:666-77, 1981). Although M. avium infections in AIDS patients are treated with 3-6 different drugs, the long term prognosis is still poor (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intern. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991).
Tuberculosis is a disease of worldwide significance and notoriety. At any one time, about one-third of the world is infected with M. tuberculosis resulting in eight million new cases of tuberculosis and 2.9 million deaths annually (A. Arachi, Tubercle. 72:1-6, 1991). It is estimated that about 0.3% of U.S. residents are infected and at risk to develop active disease (CDC 1996, CDC Revises HIV Infection Estimates. HIV/AIDS Prevention. August:2). This risk becomes even greater if the person is co-infected with the human immunodeficiency virus (HIV). If so, estimates indicate that progression to tuberculosis will occur in about 30% of those cases and the risk for developing tuberculosis becomes 113 times greater (tuberculosis., N.a.p.t.c.m.-r., MMWR. 41 RR-11:1-71, 1992).
As the projected figure for HIV infections is more than 20 million by the year 2000, it is probable that the number of tuberculosis cases worldwide will also increase. Even in 1991, the figure for people co-infected with HIV and M. tuberculosis was estimated to be 3.1 million (J. F. Murray, Bull. Int. Union Tuberc. Lung Dis. 66:21-15, 1991). In addition, life-threatening strains of MDR-TB are appearing. Some of these strains can result in a high mortality rate (e.g. 72-89%), with death occurring in a short period (e.g. 4-16 weeks) (CDC, Mortal. Morbid. Weekly Rep. 39:718-22, 1990; CDC, Mortal. Morbid. Weekly Rep. 40:649-652, 1991; B. R. Edlin et al., New Engl. J. Med. 326:1514-21, 1992). In summary, the impact of tuberculosis on the world today can best be appreciated by the fact that the World Health Organization declared tuberculosis a global public health emergency, a distinction never before given to any other disease (WHO., Soz Praventivmed. 38:251-52, 1993). Consequently, the development of new antimycobacterial drugs is an important research endeavor.
The dihydrofolate reductase (DHFR) enzyme is an important target for medicinal chemistry (K. Bowden et al., J. Chemother. 5:377-88, 1993) and DHFR inhibitors have been used in anticancer therapy (e.g. methotrexate (W. A. Bleyer, Cancer Treat. Rev. 41:36-51, 1978)), antibacterial therapy (e.g. trimethoprim (M. Finland et al., J. Infect. Dis. 128:S425-816, 1973)), and antimalarial therapy (e.g. pyrimethamine (A. K. Saxena, Prog. Drug Res. 30:221-80, 1986)). Dihydrofolate reductase is present in all cells and is necessary for the maintenance of intracellular folate pools in a biochemically active reduced state (M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991). Inhibition of the enzyme is effective because binding affinities for substrate analogs are so great that such analogs are not readily displaced by the natural substrates. Enzyme inhibition results in the depletion of intracellular reduced folates that are required for one carbon transfer reactions, which in turn are important for the biosynthesis of thymidylate, purine nucleotides, methionine, serine, glycine and many other compounds needed for RNA, DNA, and protein synthesis. FIG. 1 depicts DHFR's role in the biosynthesis of tetrahydrofolate and cell metabolism. (P. G. Hartman, J. Chemother. 5:369-76, 1993). Some bacteria have an uptake system for folates, but most have to synthesize folates de novo by reduction of dihydrofolate to tetrahydrofolates.
Although DHFR is not a new drug target, enthusiasm in the development of improved derivatives to inhibit DHFR is very intense, (D. P. Baccanari et al., J. Chemother. 5:393-99, 1993; K. Bowden et al., J. Chemother. 5:377-88, 1993; M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991; J. R. Piper et al., J. Med. Chem. 39:1271-80, 1996; B. I. Schweitzer et al., FASEB. 4:2441-52, 1990; J. K. Seydel, J. Chemother. 5:422-29, 1993), and particularly with regard to mycobacteria (K. H. Czaplinski et al., Eur. J. Med. Chem. 30:779-87, 1995; M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; H. H. Locher et al., Antimicrob. Agents Chemother. 40:1376-81, 1996; S. C. C. Meyer et al., Antimicro. Agents Chemother. 39:1862-63, 1995; R. L. Then, J. Chemother. 5:361-68, 1993). A unique feature of DHFR is the selectivity possible in the design of inhibitors for this target, thus making it an ideal target for antimycobacterial agents using rational and effective drug design. Although genes for DHFR (fol A) have been identified in other bacteria, they are not equivalent to the fol A gene from M. avium or other mycobacteria. The enzyme product of the M. avium gene (i.e. DHFR) is structurally different from other known DHFRs. For these reasons and others having to do with, for example, toxicity, selective drugs can be designed for individual species. Thus, a selective drug for M. avium can be designed in such a way that it will not affect DHFRs in other species, such as humans. One example of the species specific nature of the enzyme is demonstrated by the fact that the diaminopyrimidines, when properly substituted, can be several thousand times more active against bacterial than mammalian DHFR (P. G. Hartman, J. Chemother. 5:369-76, 1993). In addition, there are many possible inhibitors of this enzyme that have not been synthesized or studied (K. Bowden et al., J. Chemother. 5:377-88, 1993). DHFR also represents an enzyme that has been extensively used in the development of site directed inhibitors based upon X-ray crystallographic and molecular graphic studies (K. Bowden et al., J. Chemother. 5:377-388, 1993; M. P. Bradley, J. Med. Chem. 36:3171-77, 1993; B. J. Denny et al., J. Med. Chem. 35:2315-20, 1992; M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991; B. Roth, FASEB. 45:2765-72, 1986; W. M. Southerland, J. Computer-Aided Molecular Design. 8:113-22, 1994).
An important objective in the future development of DHFR inhibitors will be improving delivery of antimycobacterial drugs. One research program, designed to develop sustained and targeted delivery of first-line antituberculosis drugs using micro-encapsulation techniques has successfully formulated a micro-encapsulated form of rifampicin that shows good release characteristics (W. W. Barrow et al., European Society for Mycobacteriology, Institute Pasteur, Paris, France: 1996:50). Use of this formulation has resulted in reduction in colony forming units (CFUs) in both the M. tuberculosis H37Rv infected macrophage and mouse models, suggesting that this technology can also be used for other antimycobacterial drugs including the lipophilic DHFR inhibitors.
The history of the development of antifolates is long and includes significant contributions to that area of anticancer and anti-infective drug discovery. Improved agents against opportunistic infections have recently been developed including a synthetic process to prepare 5-alkyl-5-deaza analogs of antifolates, both classical and the lipophilic types. These analogs have been used in several studies (J. R. Piper et al., J. Med. Chem. 39:1271-80, 1996).
Mycobacterial DHFR has been a target for drug design for about three decades. Two groups in particular have synthesized lipophilic antifolates targeting this enzyme in mycobacteria. One group published activity results on a small number of 2,4-diamino-6-substituted pteridines, 6-substituted 8-deazapteridine, 5,6-substituted-5-deazapteridines and 5-methyl-6-substituted-5,8-di-deazapteridines (quinazolines) against M. species 607 (W. T. Colwell et al., Chemistry and Biology of Pteridines. vol. Elsevier North Holland, Inc., Amsterdam. 215-18, 1979; J. I. DeGraw et al., J. Medicinal Chem. 17:144-46, 1974; J. I. DeGraw et al., J. Medicinal Chem. 17:762-64, 1974). Another group published several papers describing the design and synthesis of substituted 2,4-diamino-5-benzyl pyrimidines (Trimethoprim analogs) active against mycobacterial DHFR from M. lufu and screened in vitro against M. lufu, M. tuberculosis and M. marinum (K. H. Czaplinski et al., Eur. J. Med. Chem. 30:779-87, 1995; M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; J. K. Seydel et al., Chemother. 29:249-61, 1983).
These data support the activity of folate analogs against mycobacteria. Active compounds were obtained in each series of experiments. The quinazoline analogs were targeted as lead compounds. Although quite active against isolated DHFR, these compounds demonstrated poor selectivity. However, it was apparent that selectivity was enhanced by a 5-methyl substitution for both the 5-deaza- and 5,8-di-deazapteridines (quinazolines). Although low toxicity was noted for the quinazolines in mice, equivocal results against M. leprae in the mouse foot pad model were noted (W. T. Colwell et al., Chemistry and Biology of Pteridines, Elsevier North Holland, Inc., Amsterdam. 215-18, 1979).
One research group has been pursuing trimethoprim analogs such as 2,4-diamino-5-benzylpyrimidine derivatives (M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; J. K. Seydel, J. Chemother. 5:422-29, 1993; J. K. Seydel et al., Chemother. 29:249-61, 1983). Several 4'-modified derivatives have been synthesized to extend into the glutamate binding region of the enzyme (L. F. Kuyper et al., J. Med. Chem. 28:303-11, 1985). A small number of these compounds showed very good selective activity against M. lufu in vitro and against the isolated bacterial DHFR (K. H. Czaplinski, Eur. J. Med. Chem. 30:779-87, 1995). The compounds showed an activity profile against M. tuberculosis in vitro (M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992).
The efficacy of using antifolates against M. avium and other mycobacteria has not yet been fully determined. In addition, as new antifolates are developed, their efficacy against M. avium and other mycobacteria will need to be evaluated. Such studies could be greatly facilitated through the use of purified recombinant mycobacterial DHFR.
Thus far, there has not been complete or accurate identification, sequencing and cloning of the mycobacterial DHFR gene of any species. Two papers have been published about mycobacterial DHFR. Al-Rubeai et al. (M. Al-Rubeai et al., Biochem. J. 235:301-3, 1986), reported on the purification and characterization of DHFR from M. phlei and Sirawaraporn et al. (W. Sirawaraporn et al., Exper. Parisitol. 72:184-90, 1991), reported on the purification and characterization of DHFR from a strain of M. smegmatis. The reported molecular weights were 15 and 23 kDa for M. phlei, and M. smegmatis DHFR, respectively. In Sirawaraporn et al., the authors reported on the amino terminal sequencing of the protein. Of the fifteen assignments reported, twelve were stated to be clear, one ambiguous and two could not be determined. Other than this incomplete and partial sequencing of M. segmatis DHFR protein, no mycobacterial DHFR DNA sequences have previously been reported.